Somatic cellular therapy or cell therapy, including gene therapy, already has great importance in medicine. With technological and scientific advancement the role of cell therapy and its possible applications in medicine are likely to increase.
There are currently numerous examples of work being done in the field of cell therapy. One of the most common applications is bone marrow transplantation. In this kind of cell therapy hematopoietic stem cells (HSC) are taken from a donor bone marrow, umbilical cord blood (UCB) or peripheral blood and transfused into a patient. The HSC migrate into the recipient bone marrow where some of the HSC remain as a continuous population of HSC and others differentiate into new and healthy blood cells. This treatment can be autologous, xenogeneic or allogeneic.
Examples of the types of work being done in research in cell therapy include:                Use of UCB as a source for HSC for treatment of patients whose immune systems have been damaged, such as in US patent application publication No. 2005/0020524,        Ex vivo expansion of somatic cells such as in U.S. Pat. No. 6,887,704 and US patent application publication No. 2005/0142118.        The use of somatic cells as study models for the assessment of immunotoxicity/immunotolerance, for the development of cosmetic and pharmaceutical active principles and for the development and implementation of methods of cell and tissue therapy (e.g. US patent application publication Nos. 2005/0118712, 2005/0042754, 2005/0008623, and 2005/0095228).        Culturing mesenchymal stem cells (MSC) and their preparation for therapeutic use such as in US patent application publication No. 2005/0059152. Amongst the published uses for MSC is the grafting of a donor's MSC to a recipient of an organ (from the same donor, in order to prevent graft rejection (immunosuppresion) (Ryan et al., 2005).        Use in treating circulatory disorders and heart problems such as in US patent application publication No. 2004/0197310.        The use of macrophages in wound healing (Danon et al., 1997).        
Preservation at a temperature below 0° C. (defined herein as “cryopreservation”), allows for long storage times and may be at any temperature below 0° C., including such temperatures below −20° C., −70° C., −135° C., or in liquid nitrogen. Cryopreservation is achievable by freezing or by vitrification. In vitrification, ice-crystals are not formed, however high concentrations of cryoprotectant agents that are known to be toxic must be added to the biological material. These cryoprotectant agents must be removed before the biological sample is used, in order not to harm the recipient of the biological material. Freezing is also known to cause damage. For example, ice crystals forming in the solution exert extra-cellular mechanical stress. Intracellular stress can be caused for example by osmosis of water into the extra-cellular space, to replace water that is already frozen.
One factor that has a major effect on the success of cryopreservation is the composition of the solution in which the biological material is immersed prior to freezing. Currently many different cryopreservation solutions are known. Normally, such solutions contain a balanced salt solution such as phosphate buffered saline (PBS), cryoprotectant agents (CPAs), and other molecules including butandiol and methanol.
In addition, sugars, proteins, carbohydrates such as hydroxy ethyl starch (HES), dextran, proteins (especially serum proteins such as albumin) and other macromolecules are also used and are generally termed herein “cryoprotectants”. Trehalose, for example, is thought to be protective by binding to lipid polar groups and replacing water. An example for a currently used storage technique may be found in International patent application publication No. WO 01/23532. Currently used cryoprotectant agents, namely, DMSO, ethylene glycol, glycerol and other polyalcohols, are toxic to cells and therefore upon thawing need to be removed or even washed. Use of serum may also be hazardous, since there is a hazard of contamination (especially when the source is human) or if the source is non-human (e.g. bovine) there are also health hazard (e.g. prions and ill match of the cells to the human body).
In most cryopreservation protocols, preservation of the frozen biological material is at a temperature below −130° C. This is normally done in containers of liquid nitrogen (LN) by either immersion of the biological material in LN or in LN vapor. This adds significantly to the cost of long-term preservation. In addition, incidents are known where the LN in the container evaporated (either due to a malfunction of the container or human error) and the biological materials were damaged. Furthermore, when storing in LN cross contamination can occur. This might be discovered only after use and cost also in patients' lives.
Moreover, in many applications, the cell therapy technique includes a step of processing the cells and usually such processed or treated cells are more sensitive to preservation in cold temperatures. The result is that the cells have a very short shelf life after the process is completed and the cells must be administered to a patient (e.g. injected) or otherwise used in a very short time, sometimes even a few hours after the process is completed. This limitation causes the market of providing treated or processed cells to be considered as a “service”. A good long term preservation method will allow the treated cells to be regarded more as a “product”, ready to be used whenever required and not only when their processing is completed.
One method that can overcome these obstacles is lyophilization of the frozen biological material (e.g. as described in US patent application publication No. 2004-191754). Lyophilization is a process in which ice crystals are removed by sublimation and desorption, resulting in dry, or partially dry, matter. The lyophilized material may be stored at room temperature for a long period of time and be rehydrated for use by simply adding water. Lyophilization results in higher survival rates than air drying or heating, but is still a damaging process.
In order to enhance the biological material's ability to survive the freeze-drying process, intercellular and/or extra-cellular lyoprotectant agents (LPAs) are often added to the biological material. Carbohydrates and polymers (such as PVP, Dextran, Hydroxy Ethyl Starch (HES), glucose, sucrose, mannose, lactose, trehalose and other) are known to be used for stabilization of the cells during lyophilization and storage in the dry state (Yu et al., 2004).
One method of lyophilization of cells includes introduction of trehalose into the cells. Trehalose is known to protect cell membranes in a dry state (Chen et al., 2001). It was also shown to improve platelet survival after freeze-drying (Crowe et al., 2003).
Umbilical cord blood (UCB) is a source for hematopoietic stem cells (HSC). HSC are cells that can differentiate into all blood cells. Other sources for HSC are bone marrow and a very small amount of HSC can be found circulating in peripheral blood (as WBC). Morphologically, HSC have a round nucleus similar to the mononuclear white blood cells (lymphocytes and monocytes). They resemble lymphocytes very much, and may be slightly bigger. The method to differentiate between them is according to cell membrane antigens. HSC are normally identified by expression of the CD34 antigen. HSC (from peripheral blood, bone marrow or UCB) are given to patients whose immune systems have been damaged, e.g. due to chemotherapy and/or radiotherapy and in different diseases such as: acute and chronic leukemias, myelodysplastic syndromes, Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma, aplastic anemia, thalassemia, sickle cell anemia, neuroblastoma and more.
The current method for the preservation of somatic cells is using 5-10% DMSO and storage in liquid nitrogen (LN). When storing SC from UCB and from peripheral blood the cells are separated using ficoll-paque and the fraction that is stored are the MNC (Cord Blood Bank Standard Operating Procedures, Chapter 4).
Lyophilization of mononuclear cells derived from human umbilical cord blood was described by Xiao, H. H., et al., 2004 and Lil et al. 2005.
Epigallocatechin Gallate (EGCG)
Epigallocatechin gallate (EGCG) is a polyphenol (MW 458.4) found naturally for example in green and black tea. The well-known beneficial effects associated with such tea are attributed, at least in part, to EGCG. Among the mechanisms associated with EGCG's beneficial effects are its ability to function as an antioxidant, its ability to associate with the phospholipids bi-layer of the cell membrane (Fujiki et al. 1999) and the lipid head groups of liposomes (Kumazawa et al., 2004) and more. Whilst EGCG is the main constituent of green tea, other polyphenols that are found naturally in green tea, such as epicatechin gallate (ECG) epigallocatechin (EGC) and epicatechin (EC), are also found in green tea and, like EGCG, are considered to be non-toxic. These polyphenols share structural and functional properties with EGCG (Suganuma et al. 1999).
International patent application Publication No WO02/01952 describes a preservation fluid for cells and tissues, containing a polyphenol as the active ingredient. The fluid may further contain trehalose.